Carrier molecules

ABSTRACT

The present invention relates generally to carrier molecules derived from one animal or avian species or strains and which are substantially non-immunogenic when exposed to an immune system from a species or strain of another animal or avian creature. More particularly, the present invention provides deimmunized immunointeractive molecules and even more particular deimmunized antibodies for use in diagnostic and therapeutic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/432,223, filedMay 10, 2006, which is a continuation of U.S. Ser. No. 10/184,300, filedJun. 26, 2002, now U.S. Pat. No. 7,087,724, from which applicationspriority is claimed under 35 U.S.C. §120. U.S. Ser. No. 10/184,300claims the benefit under 35 U.S.C. §119(e)(1) of U.S. Provisional No.60/301,154, filed Jun. 26, 2001 and U.S. Provisional No. 60/300,947,filed Jun. 27, 2001. The foregoing applications are incorporated hereinby reference in their entireties.

The present invention relates generally to carrier molecules derivedfrom one animal or avian species or strains and which are substantiallynon-immunogenic when exposed to an immune system from a species orstrain of another animal or avian creature. More particularly, thepresent invention provides deimmunized immunointeractive molecules andeven more particular deimmunized antibodies for use in diagnostic andtherapeutic applications.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to in thisspecification are collected at the end of the description.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Fibrinogen is a large protein molecule which normally circulates inblood plasma in a dissolved state. In the presence of thrombin,fibrinogen molecules form long thread-like polymers or networks calledfibrin which is the primary ingredient of blood clots.

Upon digestion with plasmin, fibrinogen forms fragments designated A-E.Fragments D and E are the predominant fragments and there is about twiceas much D as there is of B. Fibrinogen has a trinodular shape wherefragment E is a central component and fragment D is a terminalcomponent.

Plasmin digests of fibrin and fibrinogen can be differentiated from eachother using polyacrylamide gel electrophoresis (PAGE). Cross-linking offibrin with Factor XIIIa forms dimers of fragment D called D-dimer.Factor XIIIa is an enzyme which introduces covalent bonds betweenadjacent monomers in fibrin (Budzynski et al., Blood 54(4): 794-804,1979). Factor XIIIa is activated by the thrombin-catalyzed removal of apeptide from a precursor in plasma and in blood platelets. D-dimer is amolecule of about 189,000 daltons which consists essentially of twofragment D moieties derived from different fibrin molecules covalentlybound by cross-link bonds between the γ chain remnants of fibrinogen.Fibrinogen itself comprises six chains with two copies of an α, β and γchain.

Another complex (DD)E is formed by plasmin degradation of cross-linkedhuman fibrin and comprises a combination of two D fragments and fragmentE.

Other cross-linked derivatives are described by Graeff and Halfer(Graeff and Halfer, “Detection and Relevance of Cross-linked FibrinDerivatives in Blood”, Seminars in Thrombosis and Hemostatis 8(1), 1982)and include high molecular weight cross-linked derivatives such as DY,YY, XD, XY, DXD and YXD.

Normal haemostasis or coagulation of blood involves maintainingintravascular constituents in a liquid phase or suspension whileconcomitantly permitting local deposition of solid phase bloodcomponents in areas of vessel damage. In health, it has been assumed,but never experimentally demonstrated, that a balance exists between alow-grade intravascular deposition of fibrin and its removal byfibrinolysis or cellular phagocytosis.

Early clinical observations revealed that some severely ill patientsdeveloped signs of haemorrhage and massive bruising and had prolongedclotting times and thrombocytopenia. At postmorten, in some cases,fibrin thrombi were demonstrated in the microvasculature. The diffusenature of these thrombi gave rise to disseminated intravascularcoagulation (DIC) also known as consumptive coagulopathy. Subsequently,the thrombin were associated with conditions such as deep veinthrombosis (DVT) and pulmonary embolism (PE).

Conditions such as DIC, DVT and PE involve activation of the coagulationsystem resulting in platelet consumption, thrombin generation, fibrindeposition and secondary fibrinolysis. The net biologic effect of thisprocess reflects a balance between fibrin deposition and fibrinclearance. The resulting clinical manifestations may be haemorrhage,when depletion of coagulation factors predominates, or ischemic tissuedamage, due to the effects of vascular occlusion amongst otherconditions.

DIC, DVT and PE have been reported as a secondary phenomenon in a widevariety of disorders, particularly those accompanied by a combination ofshock, acidosis and hypoxemia. The well-recognized clinical associationsare sepsis, major trauma, malignancy and obstetric disorders. Recently,DVT has been recognized as a particular problem during prolonged airtravel or other prolonged immobility. In any event, activation of thecoagulation sequence results in consumption of coagulation protein andplatelets, leading to fibrin deposition in the micro-circulation.

Ideally, a definitive diagnosis of conditions such as DIC, DVT and PErequires the direct demonstration of diffuse fibrin deposition. Thepractical difficulty of obtaining multiple direct biopsy evidence todifferentiate between localized and generalized fibrin formation has ledto the development of indirect tests that are substituted as diagnosticend points. However, these tests are not specific for the syndrome ofintravascular fibrin deposition. Their specificity is further reduced bythe action of other enzymes that although not able to convert fibrinogento fibrin can cause similar alterations to thrombin on the othercoagulation factors involved in thrombosis. All of the indirect testsare based on the principle that thrombin is the only enzyme (snakevenoms excluded) capable of converting fibrinogen to fibrin in mammals.

Also, apart from the paracoagulation tests that detect the presence ofcirculating soluble fibrin monomer complexes, none of the more specificthrombin specific tests is readily available or useful for immediateclinical application in the diagnosis of these fibrin-associatedconditions. These tests include the FPA (fibrinopeptide A) test whereFPA is measured by a specific RIA procedure, fibrin monomer assays,fibrinogen gel exclusion chromatography and tests for FPB(fibrinopeptide B) or thrombin increasable FPB.

Tests with biochemical non-specificity for thrombin action include theprothrombin time (PT), thromboplastin time (A PTT) and thrombin clottingtime (TCT) tests. Although frequently useful in practice, it must berecognized that information obtained from these tests is non-specific innature, acting as a measure of clotting factor depletion regardless ofetiology.

Coagulation factor assays have also been found to be relativelynon-specific and these include assays for cofactors V and VIII as wellas tests for fibrinogen levels.

Tests for fibrin-fibrinogen degradation products so far have not provedto be specific for the action of plasmin on fibrin and may yieldpositive results where there has been fibrinogenolysis without priorthrombin action on the fibrinogen molecule. These tests include testsfor fragments D and E.

Tests for thrombin-mediated platelet interaction or release have beenfound to be non-specific in nature. These include platelet count,platelet survival and tests of platelet release.

The use of radio labeled fibrinogen in relation to identifying clottingfactors have also been attempted but found to be time consuming anddifficult to perform.

Thus, the efficacy of a diagnostic test lies in its ability to indicatethe presence or absence of disease. There are well recognized essentialdesign principles for studies determining the efficacy of a diagnostictest which enables the four indices of sensitivity, specificity,positive predictive value and negative predictive value to bedetermined. The first requirement is the adoption of a suitable standardfor diagnosis. Ideally, this standard should be slightly more than aclinical definition and should be as specific as possible for thedisease entity. An inherent difficulty in relation to DVT and PE inparticular is that many of the routinely available laboratory tests alsolack diagnostic specificity. A low platelet count supports thelikelihood of these conditions but may occur as an isolated findingsecondary to infection. Similar limitations apply to many of thecoagulation assays. Hypofibrinogenemia does not distinguish betweenprimary fibrinolysis, due either to the action of plasmin or elastasesand secondary fibrinolysis following the thrombin-mediated conversion offibrinogen to fibrin. Alternatively, sensitive tests of thrombin actionare available but there are obvious drawbacks with their clinical use.An example is the FPA assay which, although specific for thrombinaction, is exquisitely sensitive and may detect localized intravascularcoagulation yielding a positive result in uncomplicated venousthrombosis. The clinical significance of an elevated FPA level, evenwith a positive paracoagulation test, is then at issue, particularly ifthe platelet count, global clotting tests and fibrinogen level arenormal.

For these reasons, sensitivity, specificity and predictive values cannotbe determined in a standard fashion. The clinical presentation of thesedisorders is complex and unpredictable. The application of the availabletests for diagnosis are, therefore, best considered in relation to thedifferent clinical syndromes of intravascular coagulation.

Murine monoclonal antibody 3B6 was disclosed (U.S. Pat. No. 4,758,524).This antibody is specific for D-dimer and represents the firstclot-specific antibody. The ability to use this antibody, however, inhumans as a systemic diagnostic agent is limited due to theimmunogenicity of the molecule. There is a need, therefore, to modifythe 3B6 antibody to reduce its immunogenicity in non-murine animals andhumans.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

In work leading up to the present invention, deimmunization technologywas used to reduce the immunogenicity of the 3B6 antibody. This hasenabled the development of a thrombosis imaging diagnostic procedure foruse in humans. Furthermore, the deimmunized form of the 3B6 antibodypermits its use as a clot targeting agent to deliver clot dissolution orclot growth prevention agents such as anti-coagulants to the site of aclot. The deimmunized molecules of the present invention act, therefore,as carriers of diagnostic and therapeutic agents to a target site suchas a clot. The molecules may also have their own diagnostic ortherapeutic properties. The development of a deimmunized form of the 3B6antibody has application for a range of conditions such as DVT and PE.Furthermore, the deimmunized 3B6 antibodies can be used in combinationwith computer assisted tomographic nuclear medicine or planar imagingtechniques such as CT, MRI or ultrasound.

The present invention provides, therefore, a carrier molecule generallyin the form of an immunointeractive molecule and in particular amonoclonal antibody rendered chimeric and/or mutated relative to aparent molecule such that it exhibits reduced capacity forimmunogenicity in a target host, such as a human. The process ofchimerism or mutation is referred to herein as deimmunization. In aparticularly preferred embodiment, the immunointeractive molecule suchas the monoclonal antibody is humanized to reduce its immunogenicity inhumans. Deimmunization may be conducted in different ways but in apreferred embodiment, one or more amino acids in the variable (v) regionof a monoclonal antibody are mutated (e.g. substituted) to reduce MHC IIrecognition of peptides derived from this region. In other words, thedeimmunization process is aimed at reducing a T cell epitope-mediatedimmune response to the antibody. The most preferred antibody of thepresent invention is a deimmunized form of murine monoclonal antibody3B6 which exhibits specificity for D-dimer. The generation of adeimmunized form of 3B6 permits development inter alia of a systemicclot targeting agent for blood clots in humans. This permits its use asan imaging agent and as a vehicle to deliver clot dissolution or clotgrowth prevention agents such as to the site of a clot.

The deimmunized antibody acts, therefore, alone or as a carrier for arange of diagnostic and/or therapeutic agents.

Accordingly, one aspect of the present invention provides a variant ofan immunointeractive molecule comprising a portion having specificityfor cross-linked fibrin derivatives and which portion is derived from animmunointeractive molecule obtainable from one animal or avian creaturewherein the variant exhibits reduced immunogenicity in another animal oravian creature from the same or different species.

Preferably, the immunointeractive molecule is a variant monoclonalantibody comprising a portion having specificity for cross-linked fibrinderivatives.

More preferably, the monoclonal antibody is a variant of amurine-derived monoclonal antibody having specificity for human-derivedD-dimer and other cross-linked fibrin derivatives and non-reactivitywith fibrinogen or fibrinogen degradation products inclusive offragments D and E wherein the variant murine-derived monoclonal antibodyis substantially non-immunogenic in a human.

Preferably, the antibody is a deimmunized antibody molecule havingspecificity for an epitope recognized by monoclonal antibody 3B6 andcomprises at least one of the complementary determining regions (CDRs)of the variable domain derived from the 3B6 monoclonal antibody and theremaining immunoglobulin-derived parts of the deimmunized antibodymolecule are derived from an immunoglobulin or an analogue thereof fromthe host for which the antibody is to be deimmunized.

The present invention provides, therefore, a deimmunized antibodymolecule having specificity for an epitope recognized by monoclonalantibody 3B6 wherein at least one of the complementary determiningregions (CDRs) of the variable domain of said deimmunized antibody isderived from the 3B6 monoclonal antibody wherein one or more amino acidsin a variable region of said 3B6 antibody is mutated to reduce MHC classII recognition of peptides derived from this region.

The present invention further provides a variant of murine monoclonalantibody 3B6 deimmunized for use in humans comprising one or more aminoacid mutations in the v-region of the 3B6 antibody designed to eliminateor reduce peptide fragments of the v-region associating with MHC classII molecule.

The deimmunized immunointeractive molecules are useful alone or ascarriers of diagnostic and/or therapeutic agents to a target site suchas a blood clot.

Accordingly, the present invention contemplates a method for detecting ablood clot in a human patient by introducing into the patient adeimmunized form of murine monoclonal antibody 3B6 or an antigen-bindingfragment thereof labeled with a reporter molecule allowing disseminationof the labeled antibody throughout the circulatory system and thensubjecting the patient to reporter molecule-detection means to identifythe location of the antibody in a clot.

In an alternative embodiment, the deimmunized form of 3B6 is not labeledbut a second antibody having anti-immunoglobulin specificity is labeled.This antibody forms a labeled complex with the first mentioned antibody.

As a carrier, the deimmunized carrier may deliver any clot bindingmolecule to the site of a clot as well as other diagnostic ortherapeutic agents.

The present invention contemplates, therefore, the use of a deimmunizedmurine monoclonal antibody specific for D-dimer or other cross-linkedfibrin derivatives in the manufacture of clot imaging agent.

Yet another aspect of the present invention contemplates a method forfacilitating the dissolution or removal of a blood clot in a human, saidmethod comprising administering to said human a clot dissolution or clotgrowth prevention-effective amount of a variant murine-derivedmonoclonal antibody having specificity for human-derived D-dimer andother cross-linked fibrin derivatives and non-reactivity with fibrinogenor fibrinogen degradation products inclusive of fragments D and Ewherein said variant murine-derived monoclonal antibody is substantiallynon-immunogenic in a human wherein said monoclonal antibody furthercomprises a clot dissolution or clot growth prevention agent fused,bound or otherwise associated thereto.

Still another aspect of the present invention is directed to the use ofa variant murine-derived monoclonal antibody having specificity forhuman-derived Dimer and other cross-linked fibrin derivatives andnon-reactivity with fibrinogen or fibrinogen degradation productsinclusive of fragments D and E wherein said variant murine-derivedmonoclonal antibody is substantially non-immunogenic in a human and saidantibody further comprising a clot dissolution or clot growth preventionagent fused, bound or otherwise attached thereto in the manufacture of amedicament for the dissolution of a blot clot in a human.

A preferred molecule is a variant murine monoclonal antibody 3B6deimmunized for use in humans and comprising a combination of heavy andlight chain v-regions comprising the amino acid sequences encoded bynucleotide sequences selected from SEQ ID NO:7/SEQ ID NO:10, SEQ IDNO:8/SEQ ID NO:10, SEQ ID NO:9/SEQ ID NO:10, SEQ ID NO:7/SEQ ID NO:12,SEQ ID NO:8/SEQ ID NO:12, SEQ ID NO:8/SEQ ID NO:11, SEQ ID NO:9/SEQ IDNO:11, SEQ ID NO:9/SEQ ID NO:12 and SEQ ID NO:7/SEQ ID NO:11 orcombinations of amino acid sequences encoded by nucleotide sequenceshaving at least 70% similarity to one or both amino acid sequences ineach of the above listed pairs or nucleotide sequences capable ofhybridizing to low stringency conditions to one or both nucleotidesequences or their complementary forms in each of the above listedpairs.

The variant 3B6 antibody deimmunized for use preferably comprises acombination of heavy and light chain v-regions comprising the amino acidsequences selected from SEQ ID NO:1/SEQ ID NO:4, SEQ ID NO:2/SEQ IDNO:4, SEQ ID NO:3/SEQ ID NO:4, SEQ ID NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQID NO:6, SEQ ID NO:2/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:5, SEQ IDNO:3/SEQ ID NO:6 and SEQ ID NO:1/SEQ ID NO:5 or combinations of aminoacid sequences having at least 70% similarity to one or both amino acidsequences in each of the above listed pairs.

Another preferred variant of 3B6 comprises a combination of heavy andlight chain v-regions selected from VHv5/VKv1, VHv6/VKv1, VHv7/VKv1,VHv5/VKv7, VHv6/VKv7, VHv6/VKv4, VHv7/VKv4, VHv7/VKv7 and VHv5/VKv4.

Another aspect of the present invention contemplates a method fordetecting a blood clot in a human patient by introducing into thepatient a deimmunized form of murine monoclonal antibody 3B6 or anantigen-binding fragment thereof labeled with a reporter moleculeallowing dissemination of the labeled antibody throughout thecirculatory system and then subjecting the patient to a computerassisted tomographic nuclear medicine scan to visualize the clot.

Yet another aspect of the present invention contemplates a method fordetecting a blood clot in a human patient by introducing into thepatient a deimmunized form of murine monoclonal antibody 3B6 or anantigen-binding fragment thereof labeled with a reporter moleculeallowing dissemination of the labeled antibody throughout thecirculatory system and then subjecting said patient to planar clotimaging to visualize the clot.

The deimmunized immunointeractive molecule of the present invention isalso useful to anchor an anti-coagulant at a particular site. Thisaspect provides, therefore, tissue-specific anchoring of a diagnostic ortherapeutic agent to, for example, a clot. Furthermore, theimmunointeractive molecule may be engineered to have multiplespecificities. For example, a bi-specific deimmunized antibody iscontemplated which comprises one specificity for a clot and anotherspecificity to a site of a clot (e.g. to a cell receptor). This enablesthe antibody to remain at the site of the clot.

In an alternative, a multi-step treatment is contemplated where, forexample, the deimmunized 3B6 or other interactive molecule conjugated toan anti-coagulant is administered to target the clot and forms a complexand then a second antibody directed to the first antibody and/or theanti-coagulant is administered to enhance or monitor the first complex.

The deimmunized immunointeracative molecule may also be used todetermine the kinetics of clot dissipation or clot disappearance. Thisis useful to predict even earlier the appearance or disappearance ofclots and, hence, aids in determining the kinetics of when to initiatesecond treatments such as anti-coagulant treatments.

The present invention further provides conjugates comprising thedeimmunized immunointeractive molecules and imaging and/or therapeutictags. Examples of imaging tags include MRI, ultrasound and CT tags.Examples of therapeutic tags include radioactive isotopes, anti-clottingagents and cytokines.

A summary of sequence identifiers used throughout the subjectspecification is provided in Table 1.

TABLE 1 Summary of Sequence Identifiers SEQUENCE ID NO: DESCRIPTION 1Amino acid of 3B6DIVHv5 2 Amino acid of 3B6DIVHv6 3 Amino acid of3B6DIVHv7 4 Amino acid of 3B6DIVKv1 5 Amino acid of 3B6DIVKv4 6 Aminoacid 3B6DIVKv7 7 Nucleotide sequence encoding 3B6DIVHv5 8 Nucleotidesequence encoding 3B6DIVHv6 9 Nucleotide sequence encoding 3B6DIVHv7 10Nucleotide sequence encoding 3B6DIVKv1 11 Nucleotide sequence encoding3B6DIVKv4 12 Nucleotide sequence encoding 3B6DIVKv7

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a representation showing (A) a schematic of the 3B6 monoclonalantibody; (B) a photograph of 3B6 binding to blood clots (×4,2000magnification).

FIG. 2 is a schematic representation of a 3B6 antibody labeled with anuclear tag (^(99m)Tc).

FIG. 3A is a diagrammatic representation showing administration of 3B6^(99m)Tc to the circulatory system of a human.

FIG. 3B is a photographic representation showing visualization of bloodclots in the anterior thighs as radiation from ^(99m)Tc concentrates atthe clot site.

FIGS. 4A, 4B and 4C are graphical representations showing D-dimercapture assay using deimmunized 3B6 monoclonal antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated in part on the application ofbiochemical techniques to render an immunointeractive molecule derivedfrom one animal or avian creature substantially non-immunogenic inanother animal or avian creature of the same or different species. Thebiochemical process is referred to herein as “deimmunization”. Referenceherein to “deimmunization” includes processes such as complementarydeterminant region (CDR) grafting, “reshaping” with respect to aframework region of an immunointeractive molecule and variable (v)region mutation, all aimed at reducing the immunogenicity of animmunointeractive molecule in a particular host. In the present case,the preferred immunointeractive molecule is an antibody such as apolyclonal or monoclonal antibody. In a most preferred embodiment, theimmunointeractive molecule is a monoclonal antibody, derived from oneanimal or avian creature and which exhibits reduced immunogenicity inanother animal or avian creature from the same or different species.

The present invention relates generally to carrier molecules derivedfrom one animal or avian species or strains and which are substantiallynon-immunogenic when exposed to an immune system from a species orstrain of another animal or avian creature. The carrier molecules mayexhibit useful diagnostic or therapeutic properties per se or may beused to deliver active compounds (e.g. anti-clotting agents, radioactiveisotopes) to a target site. Generally, the carrier molecules areimmunointeractive molecules. More particularly, the present invention isdirected to a deimmunized including a non-murine mammalianized form of amurine-derived monoclonal antibody substantially incapable of inducingan immune response against itself in a non-murine animal and inparticular a human. Even more particularly, the present inventionprovides a deimmunized form of murine monoclonal antibody 3B6 such thatit is incapable of, or exhibits reduced capacity for, inducing asubstantial immune response against itself or its derivatives whenadministered to a human. The deimmunized immunointeractive molecules andin particular antibodies of the present invention have a range of usefuldiagnostic and therapeutic applications such as in the detection ofblood clots in the circulatory system of a human and as a clot targetingagent to deliver a clot dissolution or clot growth prevention agent suchas an anticoagulant. The demmunized including humanized forms of thesubject monoclonal antibody are particularly useful in the diagnosis andtreatment of conditions such as deep vein thrombosis and pulmonaryembolism. The molecules of the present invention are particularly usefulas agents for delivering active compounds to a target site. Such activecompounds include clot binding molecules.

Accordingly, one aspect of the present invention provides a variant ofan immunointeractive molecule, said variant comprising a portion havingspecificity for cross-linked fibrin derivatives and which portion isderived from an immunointeractive molecule obtainable from one animal oravian creature wherein said variant exhibits reduced immunogenicity inanother animal or avian creature from the same or different species.

As stated above, the preferred form of immunointeractive molecule is anantibody and in particular a monoclonal antibody.

Accordingly, another aspect of the present invention provides a variantmonoclonal antibody comprising a portion having specificity forcross-linked fibrin derivatives and which portion is derived from amonoclonal antibody obtainable from a first animal or avian creaturewherein said variant exhibits reduced immunogenicity in a second animalor avian creature from the same or different species.

In a particularly preferred embodiment, a monoclonal antibody isobtained from a marine animal and is deimmunized with respect to anothermurine animal or a different species of animal such as a human. Themurine monoclonal antibody is raised in the murine animal tonon-denatured D-dimer which is derived from fibrinogen. The lattermolecule is digested by plasmin and generates a range of fragmentsdesignated fragments A to E. Cross-linking of fibrin with Factor XIIIaforms dimers of fragment D referred to as “D-dimer”. The D-dimer is amolecule of about 189,000 daltons and comprises two fragment D moietiesbound by cross-linked bonds between the γ chain remnants of fibrinogen.

Accordingly, another aspect of the present invention is directed to avariant murine-derived monoclonal antibody having specificity forhuman-derived D-dimer and other cross-linked fibrin derivatives andnon-reactivity with fibrinogen or fibrinogen degradation productsinclusive of fragments D and E wherein said variant murine-derivedmonoclonal antibody is substantially non-immunogenic in a human.

Reference to “substantially non-immunogenic” includes reducedimmunogenicity compared to a parent antibody, i.e. an antibody beforeexposure to deimmunization processes. The term “immunogenicity” includesan ability to provoke, induce or otherwise facilitate a humoral and/orT-cell mediated response in a host animal. Particularly convenientimmunogenic criteria include the ability for amino acid sequencesderived from a variable (v) region of an antibody to interact with MHCclass II molecules thereby stimulating or facilitating a T-cellmediating response including a T-cell-assisted humoral response. Theimmunointeractive molecule and in particular a monoclonal antibodycontemplated by the present invention includes reference to a clottargeting agent.

The preferred murine-derived monoclonal antibody is referred to hereinas monoclonal antibody 3B6 which is described in U.S. Pat. No.4,758,524.

Accordingly, in a particularly preferred embodiment, the presentinvention provides a deimmunized form of monoclonal antibody 3B6 whereinsaid deimmunized 3B6 is substantially non-immunogenic in humans.

Again, “substantially non-immunogenic” in this context means a reducedcapacity of the deimmunized 3B6 monoclonal antibody to induce orfacilitate an immune response against itself (following initial orsubsequent administration) in a human compared to murine monoclonalantibody 3B6, prior to deimmunization.

Although the preferred invention is particularly directed to adeimmunized form of 3B6 with respect to humans, the present inventionextends to this antibody or another antibody with a similar specificityfor D-dimer and/or other cross-linked fibrin derivatives deimmunized forany other animal or avian species.

Reference herein to other cross-linked fibrin derivatives includes, forexample, in addition to D-dimer, derivatives of D-dimer and a complexcomprising D and E fragments. The latter includes (DD)E and is formed byplasmin degradation of cross-linked human fibrin and comprises acombination of two D fragments and fragment E. Other cross-linkedderivatives include DY, YY, XD, XY, DXD and YXD where the lettersrepresent fragments of fibrinogen formed following degradation byplasmin wherein X and Y are different and are selected from fragments Ato C and E. Preferably, the deimmunized antibody of the subjectinvention is derived from an antibody specific for D-dimer and othercross-linked fibrin derivatives but which does not cross-react withfibrinogen, fibrinogen degradation products inclusive of fragment D andfragment E. Preferably, the antibody-producing clones are selected usingsolution phase D-dimer molecules rather than immobilized D-dimeralthough clones selected by either form of D-dimer are contemplated bythe present invention.

Preferably, the deimmunized antibody exhibits an affinity for its targetantigen which is similar to the affinity exhibited by murine monoclonalantibody 3B6.

By “affinity” in relation to the interaction between an individualantigen binding site on an antigen-binding molecule and itscorresponding site on the antigen includes the strength of thisinteraction.

By “antibody” is meant a protein of the immunoglobulin family that iscapable of combining, interacting or otherwise associating with anantigen. An antibody is, therefore, an antigen-binding molecule. An“antibody” is an example of an immunointeractive molecule and includes apolyclonal or monoclonal antibody. The preferred immunointeractivemolecules of the present invention are monoclonal antibodies. Anantibody includes parts thereof including Fab portions andantigen-binding determinants.

The term “antigen” is used herein in its broadest sense to refer to asubstance that is capable of reacting in and/or inducing an immuneresponse. Reference to an “antigen” includes an antigenic determinant orepitope. The antigen in the present context is regarded as theimmunointeractive molecule and, more particularly, a monoclonalantibody.

Any molecule that has binding affinity for a target antigen is referredto as an “antigen-binding molecule”. It will be understood that thisterm extends to immunoglobulins (e.g. polyclonal or monoclonalantibodies), immunoglobulin fragments and non-immunoglobulin derivedprotein frameworks that exhibit antigen-binding activity. The terms“antibody” and “antigen-binding molecules” include deimmunized forms ofthese molecules.

That part of an antigenic molecule against which a particular immuneresponse is directed is referred to as an “antigenic determinant” or“epitope” and includes a hapten. Typically, in an animal, antigenspresent several or even many antigenic determinants simultaneously. A“hapten” is a substance that can combine specificity with an antibodybut cannot or only poorly induces an immune response unless bound to acarrier. A hapten typically comprises a single antigenic determinant orepitope.

As stated above, although the preferred antibodies of the presentinvention are deimmunized forms of murine monoclonal antibodies for usein humans, the subject invention extends to antibodies from any sourceand deimmunized for use in any host. Examples of animal and aviansources and hosts include humans, primates, livestock animals (e.g.sheep, cows, horses, pigs, donkeys), laboratory test animals (e.g. mice,rabbits, guinea pigs, hamsters), companion animals (e.g. dogs, cats),poultry bird (e.g. chickens, ducks, geese, turkeys) and game birds (e.g.pheasants). The deimmunized antibodies or part thereof may also begenerated in non-animal tissues such as plants. Plants are particularlyuseful as a source of single chain antibodies.

Another aspect of the present invention contemplates a method forgenerating a deimmunized monoclonal antibody having specificity forantigenic determinants on human D-dimer or other cross-linked fibrinderivatives, said method comprising:

(i) obtaining a cross-linked fibrin derivative or extract containingsame from a human;

(ii) generating an antibody in a non-human animal specific to saidcross-linked fibrin derivative but which does not cross-react withfragment D; and

(iii) subjecting said non-human derived antibody to deimmunizationmeans.

The cross-linked fibrin derivative may be derived from any suitableantigenic extract including plasmin-mediated degradation of fibrin clotsor by simultaneous action of thrombin, Factor XIIIa and plasmin onfibrinogen with transient clot formation and subsequent clot lysis. Inthe latter method, the fibrinogen is converted to fibrin by the actionof thrombin and Factor XIIIa and subsequently digested with plasmin. Itwill, of course, be appreciated that the fibrin derivative or extractcontaining same may be obtained from an animal source other than human.The antigenic source is conveniently from a biological sample.

A sample that may be extracted, untreated, treated, diluted orconcentrated from an animal is included in the term “biological sample”.

The above method of obtaining the crude antigenic fraction represents anin vitro method. A suitable in vivo method includes obtaining sera orother body fluid containing the cross-linked fibrin derivative from ananimal including human and subjecting the body fluid to a PAGE processwherein substantially pure cross-linked fibrin derivative is isolated.

Alternatively, cross-linked fibrin derivatives may be purified fromserum obtained from patients suffering severe thrombotic disorders basedon a technique using gel filtration in combination with ion exchangechromatography as described by Willner et al., Biochemistry 21:2687-2692, 1982.

The antigen (i.e. D-dimer or other cross-linked fibrin derivative) canbe separated from the biological sample by any suitable means. Forexample, the separation may take advantage of any one or more of theantigen's surface charge properties, size, density, biological activityand its affinity for another entity (e.g. another protein or chemicalcompound to which it binds or otherwise associates). Thus, for example,separation of the antigen from the biological fluid may be achieved byany one or more of ultra-centrifugation, ion-exchange chromatography(e.g. anion exchange chromatography, cation exchange chromatography),electrophoresis (e.g. polyacrylamide gel electrophoresis, isoelectricfocussing), size separation (e.g., gel filtration, ultra-filtration) andaffinity-mediated separation (e.g. immunoaffinity separation including,but not limited to, magnetic bead separation such as Dynabead™separation, immunochromatography, immuno-precipitation). Choice of theseparation technique(s) employed may depend on the biological activityor physical properties of the particular antigen.

Preferably, the separation of the antigen from the biological fluidpreserves conformational epitopes present on the antigen surface and,thus, suitably avoids techniques that cause denaturation of the antigen.Persons of skill in the art will recognize the importance of maintainingor mimicking as close as possible physiological conditions peculiar tothe antigen (e.g. the biological fluid from which they are obtained) toensure that the antigenic determinants or active site/s on the antigen,which are exposed to the animal, are structurally identical to that ofthe native antigen. This ensures the raising of appropriate antibodiesin the immunised animal that would recognize the native antigen. In apreferred embodiment of this type, the antigen is separated from thebiological fluid using any one or more of affinity separation, gelfiltration and ultra-filtration.

Immunization and subsequent production of monoclonal antibodies can becarried out using standard protocols as for example described by Köhlerand Milstein (Nature 256: 495-499, 1975; Köhler and Milstein, Eur. J.Immunol. 6(7): 511-519, 1976), Coligan et al. (Current Protocols inImmunology, John Wiley & Sons, Inc., 1991-1997) or Toyama et al.(“Monoclonal Antibody, Experiment Manual”, published by KodanshaScientific, 1987). Essentially, an animal is immunized with anantigen-containing biological fluid or fraction thereof by standardmethods to produce antibody-producing cells, particularlyantibody-producing somatic cells (e.g. B lymphocytes). These cells canthen be removed from the immunized animal for immortalization. Theantigen may need to first be associated with a larger molecule. Thelatter is any substance of typically high molecular weight to which anon- or poorly immunogenic substance (e.g. a hapten) is naturally orartificially linked to enhance its immunogenicity.

Immortalization of antibody-producing cells may be carried out usingmethods, which are well-known in the art. For example, theimmortalization may be achieved by the transformation method usingEpstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology 121: 140,1986). In a preferred embodiment, antibody-producing cells areimmortalized using the cell fusion method (described in Coligan et al.,1991-1997, supra), which is widely employed for the production ofmonoclonal antibodies. In this method, somatic antibody-producing cellswith the potential to produce antibodies, particularly B cells, arefused with a myeloma cell line. These somatic cells may be derived fromthe lymph nodes, spleens and peripheral blood of primed animals,preferably rodent animals such as mice and rats. In the exemplaryembodiment of this invention mice, spleen cells are used. It would bepossible, however, to use rat, rabbit, sheep or goat cells, or cellsfrom other animal species instead.

Specialized myeloma cell lines have been developed from lymphocytictumours for use in hybridoma-producing fusion procedures (Köhler andMilstein, 1976, supra; Shulman et al., Nature 276: 269-270, 1978; Volket al., J. Virol. 42(1): 220-227, 1982). These cell lines have beendeveloped for at least three reasons. The first is to facilitate theselection of fused hybridomas from unfused and similarly indefinitelyself-propagating myeloma cells. Usually, this is accomplished by usingmyelomas with enzyme deficiencies that render them incapable of growingin certain selective media that support the growth of hybridomas. Thesecond reason arises from the inherent ability of lymphocytic tumourcells to produce their own antibodies. To eliminate the production oftumour cell antibodies by the hybridomas, myeloma cell lines incapableof producing endogenous light or heavy immunoglobulin chains are used. Athird reason for selection of these cell lines is for their suitabilityand efficiency for fusion.

Many myeloma cell lines may be used for the production of fused cellhybrids, including, e.g. P3X63-Ag8, P3X63-AG8.653, P3/NS1-Ag4-1 (NS-1),Sp2/0-Ag14 and S194/5.XXO.Bu.1. The P3X63-Ag8 and NS-1 cell lines havebeen described by Köhler and Milstein (1976, supra). Shulman et al.(1978, supra) developed the Sp2/0-Ag14 myeloma line. The S194/5.XXO.Bu.1line was reported by Trowbridge (J. Exp. Med. 148(1): 313-323, 1978).

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually involve mixing somatic cells withmyeloma cells in a 10:1 proportion (although the proportion may varyfrom about 20:1 to about 1:1), respectively, in the presence of an agentor agents (chemical, viral or electrical) that promotes the fusion ofcell membranes. Fusion methods have been described (Köhler and Milstein,1975, supra; Köhler and Milstein, 1976, supra; Gefter et al., SomaticCell Genet. 3: 231-236, 1977; Volk et al., 1982, supra). Thefusion-promoting agents used by those investigators were Sendai virusand polyethylene glycol (PEG).

Because fusion procedures produce viable hybrids at very low frequency(e.g. when spleens are used as a source of somatic cells, only onehybrid is obtained for roughly every 1×10⁵ spleen cells), it ispreferable to have a means of selecting the fused cell hybrids from theremaining unfused cells, particularly the unfused myeloma cells. A meansof detecting the desired antibody-producing hybridomas among otherresulting fused cell hybrids is also necessary. Generally, the selectionof fused cell hybrids is accomplished by culturing the cells in mediathat support the growth of hybridomas but prevent the growth of theunfused myeloma cells, which normally would go on dividing indefinitely.The-somatic cells used in the fusion do not maintain long-term viabilityin in vitro culture and hence do not pose a problem. In the example ofthe present invention, myeloma cells lacking hypoxanthine phosphoribosyltransferase (HPRT-negative) were used. Selection against these cells ismade in hypoxanthine/aminopterin/thymidine (HAT) medium, a medium inwhich the fused cell hybrids survive due to the HPRT-positive genotypeof the spleen cells. The use of myeloma cells with different geneticdeficiencies (drug sensitivities, etc.) that can be selected against inmedia supporting the growth of genotypically competent hybrids is alsopossible.

Several weeks are required to selectively culture the fused cellhybrids. Early in this time period, it is necessary to identify thosehybrids which produce the desired antibody, so that they maysubsequently be cloned and propagated. Generally, around 10% of thehybrids obtained produce the desired antibody, although a range of fromabout 1 to about 30% is not uncommon. The detection ofantibody-producing hybrids can be achieved by any one of severalstandard assay methods, including enzyme-linked immunoassay andradioimmunoassay techniques as, for example, described in Kennet et al.((eds) Monoclonal Antibodies and Hybridomas: A New Dimension inBiological Analyses, pp. 376-384, Plenum Press, New York, 1980). In aparticularly preferred embodiment, an enyme linked immunosorbent assay(ELISA) is performed to selected antibody producing clones usingsolution phase D-dimer.

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line may bepropagated in either of two standard ways. A suspension of the hybridomacells can be injected into a histocompatible animal. The injected animalwill then develop tumours that secrete the specific monoclonal antibodyproduced by the fused cell hybrid. The body fluids of the animal, suchas serum or ascites fluid, can be tapped to provide monoclonalantibodies in high concentration. Alternatively, the individual celllines may be propagated in vitro in laboratory culture vessels. Theculture medium containing high concentrations of a single specificmonoclonal antibody can be harvested by decantation, filtration orcentrifugation, and subsequently purified.

The cell lines are tested for their specificity to detect the antigen ofinterest by any suitable immunodetection means. For example, cell linescan be aliquoted into a number of wells and incubated and thesupernatant from each well is analyzed by enzyme-linked immunosorbentassay (ELISA), indirect fluorescent antibody technique, or the like. Thecell line(s) producing a monoclonal antibody capable of recognizing thetarget antigen but which does not recognize non-target epitopes areidentified and then directly cultured in vitro or injected into ahistocompatible animal to form tumours and to produce, collect andpurify the required antibodies.

Thus, the present invention provides in a first step monoclonalantibodies which specifically interact with D-dimer or othercross-linked fibrin derivative.

As indicated above, non-animal cells such as a plant, yeast and/ormicrobial cells may be used to generate typically single-chainantibodies. In this embodiment, such cells are engineered to expressnucleic acid molecules which encode a chain of an antibody.

The monoclonal antibody is then subjected to deimmunization means. Sucha process may take any of a number of forms including the preparation ofchimeric antibodies which have the same or similar specificity as themonoclonal antibodies prepared according to the present invention.Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin variable and constant region genes belonging to differentspecies. Thus, in accordance with the present invention, once ahybridoma producing the desired monoclonal antibody is obtained,techniques are used to produce interspecific monoclonal antibodieswherein the binding region of one species is combined with a non-bindingregion of the antibody of another species (Liu et al., Proc. Natl. Acad.Sci. USA 84: 3439-3443, 1987). For example, the CDRs from a non-human(e.g. murine) monoclonal antibody can be grafted onto a human antibody,thereby “humanizing” the murine antibody (European Patent PublicationNo. 0 239 400; Jones et al., Nature 321: 522-525, 1986; Verhoeyen etal., Science 239: 1534-1536, 1988; Riechmann et al., Nature 332:323-327, 1988). In this case, the deimmunizing process is specific forhumans. More particularly, the CDRs can be grafted onto a human antibodyvariable region with or without human constant regions. The non-humanantibody providing the CDRs is typically referred to as the “donor” andthe human antibody providing the framework is typically referred to asthe “acceptor”. Constant regions need not be present, but if they are,they must be substantially identical to human immunoglobulin constantregions, i.e. at least about 85-90%, preferably about 95% or moreidentical. Hence, all parts of a humanized antibody, except possibly theCDRs, are substantially identical to corresponding parts of naturalhuman immunoglobulin sequences. Thus, a “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. A donor antibody is said to be “humanized”, by theprocess of “humanization”, because the resultant humanized antibody isexpected to bind to the same antigen as the donor antibody that providesthe CDRs. Reference herein to “humanized” includes reference to anantibody deimmunized to a particular host, in this case, a human host.

It will be understood that the deimmunized antibodies may haveadditional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. Exemplary conservative substitutions may be made according toTable 2.

TABLE 2 ORIGINAL EXEMPLARY RESIDUE SUBSTITUTIONS Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Exemplary methods which may be employed to produce deimmunizedantibodies according to the present invention are described, forexample, in Richmann et al., 1988, supra; U.S. Pat. Nos. 6,056,957,6,180,370 and 6,180,377 and Chothia et al., J. Mol. Biol. 196: 901,1987.

Thus, in one embodiment, the present invention contemplates adeimmunized antibody molecule having specificity for an epitoperecognized by monoclonal antibody 3B6 wherein at least one or at leasttwo or at least three or at least four or at least five of thecomplementary determining regions (CDRs) of the variable domain of saiddeimmunized antibody is derived from said 3B6 monoclonal antibody andthe remaining immunoglobulin-derived parts of the deimmunized antibodymolecule are derived from an immunoglobulin or an analogue thereof fromthe host for which the antibody is to be deimmunized.

This aspect of the present invention involves manipulation of theframework region of a non-human antibody.

Preferably, the deimmunized antibody is a humanized form of murine 3B6.

One preferred deimmunization process is referred to herein avariable(v)-region grafting and results in a chimeric antibody. Theresulting antibody comprises one or more amino acid substitutions withinthe v-region when compared to the present (e.g. murine) antibody. Therationale for making v-region changes is to further the potential for aninduced immune response in the intended host (e.g. a human). The basisof deimmunization is predicated in part on the assumption that asubstantive immune response to an introduced antibody requires a T-cellmediated response. The trigger for the T-cell response is thepresentation of processed peptides emanating from the introducedantibody on the surface of antigen presenting cells (APCs). The APCspresent such peptides in association with surface MHC class IImolecules. The deimmunized approach is, therefore, based on:

(i) predicting peptide sequences capable of associating with MHC classII molecules; and

(ii) changing strategic residues to eliminate the ability of the peptideto associate with the MHC class II molecule.

Accordingly, another aspect of the present invention provides a variantof murine monoclonal antibody 3B6 deimmunized for use in humans, saidvariant comprising one or more amino acid mutations in the v-region ofsaid 3B6 antibody to eliminate or reduce peptide fragments of saidv-region associating with MHC class II molecules.

One or more amino acid substitutions, additions and/or deletions or oneor more nucleotide substitutions, additions and/or deletions isencompassed by the term “mutation” or “mutations”.

In a particularly preferred embodiment, the deimmunized antibodies aregenerated by co-transfection of different combinations of threedeimmunized H-chain genes and three deimmunized L-chain genes. Theresulting variants are derived from different combinations encoded byH-chain and L-chain genes. Preferred H-chains are Hv5, Hv6 and Hv7.These are referred to herein as 3B6DIVHv5 (SEQ ID NO:1), 3B6DIVHv6 (SEQID NO:2) and 3B6DIVHv7 (SEQ ID NO:3). Preferred L-chains are Kv1, Kv4and Kv7. These are referred to herein as 3B6DIVKv1 (SEQ ID NO:4),3B6DIVKv4 (SEQ ID NO:5) and 3B6DIVKv7 (SEQ ID NO:6). Particularly usefulcombinations include VHv5/VKv1, VHv6/VKv1, VHv7/VKv1, VHv5/VKv7,VHv6/VKv7, VHv6/VKv4, VHv7/VKv4, VHv7/VKv7 and VHv5/VKv4. The sequenceidentifier numbers (SEQ ID NOS:) in parentheses represent the amino acidsequences of the particular chains. Corresponding nucleotide sequencesencoding each of SEQ ID NOS:1-6 are represented by SEQ ID NOS:7-12.

All such combinations of H and L chains are also encompassed by thepresent invention.

Accordingly, the present invention provides a variant murine monoclonalantibody 3B6 deimmunized for use in humans, said variant comprising acombination of heavy and light chain v-regions comprising the amino acidsequences encoded by nucleotide sequences selected from SEQ ID NO:7/SEQID NO:10, SEQ ID NO:8/SEQ ID NO:10, SEQ ID NO:9/SEQ ID NO:10, SEQ IDNO:7/SEQ ID NO:12, SEQ ID NO:8/SEQ ID NO:12, SEQ ID NO:8/SEQ ID NO:1,SEQ ID NO:9/SEQ ID NO:11, SEQ ID NO:9/SEQ ID NO:12 and SEQ ID NO:7/SEQID NO:11 or combinations of amino acid sequences encoded by nucleotidesequences having at least 70% similarity to one or both amino acidsequences in each of the above listed pairs or nucleotide sequencescapable of hybridizing to low stringency conditions to one or bothnucleotide sequences or their complementary forms in each of the abovelisted pairs.

All such combinations of H and L chains are also encompassed by thepresent invention.

Accordingly, the present invention provides a variant murine monoclonalantibody 3B6 deimmunized for use in humans, said variant comprising acombination of heavy and light chain v-regions comprising the amino acidsequences selected from SEQ ID NO:1/SEQ ID NO:4, SEQ ID NO:2/SEQ IDNO:4, SEQ ID NO:3/SEQ ID NO:4, SEQ ID NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQID NO:6, SEQ ID NO:2/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:5, SEQ IDNO:3/SEQ ID NO:6 and SEQ ID NO:1/SEQ ID NO:5 or combinations of aminoacid sequences encoded by nucleotide sequences having at least 70%similarity to one or both amino acid sequences in each of the abovelisted pairs.

More particularly, the present invention provides a variant of a murinemonoclonal antibody 3B6 deimmunized for use in humans, said variantcomprising a combination of heavy and light chain v-regions selectedfrom VHv5/VKv1, VHv6/VKv1, VHv7/VKv1, VHv5/VKv7, VHv6/VKv7, VHv6/VKv4,VHv7/VKv4, VHv7/VKv7 and VHv5/VKv4.

The term “similarity” as used herein includes exact identity betweencompared sequences at the nucleotide or amino acid level. Where there isnon-identity at the nucleotide level, “similarity” includes differencesbetween sequences which result in different amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. Where there is non-identity atthe amino acid level, “similarity” includes amino acids that arenevertheless related to each other at the structural, functional,biochemical and/or conformational levels. In a particularly preferredembodiment, nucleotide and sequence comparisons are made at the level ofidentity rather than similarity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence similarity”, “sequence identity”,“percentage of sequence similarity”, “percentage of sequence identity”,“substantially similar” and “substantial identity”. A “referencesequence” is at least 12 but frequently 15 to 18 and often at least 25or above, such as 30 monomer units, inclusive of nucleotides and aminoacid residues, in length. Because two polynucleotides may each comprise(1) a sequence (i.e. only a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) asequence that is divergent between the two polynucleotides, sequencecomparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment oftypically 12 contiguous residues that is compared to a referencesequence. The comparison window may comprise additions or deletions(i.e. gaps) of about 20% or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by computerised implementations ofalgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science DriveMadison, Wis., USA) or by inspection and the best alignment (i.e.resulting in the highest percentage homology over the comparison window)generated by any of the various methods selected. Reference also may bemade to the BLAST family of programs as, for example, disclosed byAltschul et al. (Nucl. Acids Res. 25: 3389-3402. 1997). A detaileddiscussion of sequence analysis can be found in Unit 19.3 of Ausubel etal. (“Current Protocols in Molecular Biology” John Wiley & Sons Inc,1994-1998, Chapter 15).

The terms “sequence similarity” and “sequence identity” as used hereinrefers to the extent that sequences are identical or functionally orstructurally similar on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity”, for example, is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala,Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp,Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. For the purposes of the present invention, “sequenceidentity” will be understood to mean the “match percentage” calculatedby the DNASIS computer program (Version 2.5 for windows; available fromHitachi Software engineering Co., Ltd., South San Francisco, Calif.,USA) using standard defaults as used in the reference manualaccompanying the software. Similar comments apply in relation tosequence similarity.

Mutations and derivatives contemplated by the present invention includeredundant mutations in nucleotide sequences which do not result in achange in amino acid sequence.

Reference herein to a low stringency includes and encompasses from atleast about 0 to at least about 15% v/v formamide and from at leastabout 1 M to at least about 2 M salt for hybridization, and at leastabout 1 M to at least about 2 M salt for washing conditions. Generally,low stringency is at from about 25-30° C. to about 42° C. Thetemperature may be altered and higher temperatures used to replaceformamide and/or to give alternative stringency conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization, and at least about 0.5 M to atleast about 0.9 M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.01 M to at least about 0.15M salt for hybridization, and at least about 0.01 M to at least about0.15 M salt for washing conditions. In general, washing is carried outT_(m)=69.3+0.41 (G+C) % (Mammur and Doty, J. Mol. Biol. 5: 109, 1962).However, the T_(m) of a duplex DNA decreases by 1° C. with everyincrease of 1% in the number of mismatch base pairs (Bonner and Laskey,J. Mol. Biol. 5: 109, 1962). Formamide is optional in thesehybridization conditions. Accordingly, particularly preferred levels ofstringency are defined as follows: low stringency is 6×SSC buffer, 0.1%w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/vSDS at a temperature in the range 20° C. to 65° C.; high stringency is0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.

As used herein, the term “CDR” includes CDR structural loops whichcovers to the three light chain and the three heavy chain regions in thevariable portion of an antibody framework region which bridge β strandson the binding portion of the molecule. These loops have characteristiccanonical structures (Chothia et al., J. Mol. Biol. 227: 799, 1992;Kabat et al, “Sequences of Proteins of Immunological Interest”, U.S.Department of Health and Human Services, 1983).

An immunoglobulin light or heavy chain variable region, which isinterrupted by three hypervariable regions, also called CDRs, isreferred to herein as a “framework region”. The extent of the frameworkregion and CDRs have been precisely defined (see, for example, Krebberet al., J. Immunol. Methods 201(1): 35-55, 19). The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. As used herein, a “human framework region”is a framework region that is substantially identical (about 85% ormore, usually 90-95% or more) to the framework region of a naturallyoccurring human immunoglobulin. The framework region of an antibody,that is the combined framework regions of the constituent light andheavy chains, serves to position and align the CDRs. The CDRs areprimarily responsible for binding to an epitope of an antigen.

As used herein, the term “heavy chain variable region” means apolypeptide which is from about 110 to 125 amino acid residues inlength, the amino acid sequence of which corresponds to that of a heavychain of a monoclonal antibody of the invention, starting from theamino-terminal (N-terminal) amino acid residue of the heavy chain.Likewise, the term “light chain variable region” means a polypeptidewhich is from about 95 to 130 amino acid residues in length, the aminoacid sequence of which corresponds to that of a light chain of amonoclonal antibody of the invention, starting from the N-terminal aminoacid residue of the light chain. Full-length immunoglobulin “lightchains” (about 25 Kd or 214 amino acids) are encoded by a variableregion gene at the NH₂-terminus (about 110 amino acids) and a κ or λconstant region gene at the COOH-terminus. Full-length immunoglobulin“heavy chains” (about 50 Kd or 446 amino acids), are similarly encodedby a variable region gene (about 116 amino acids) and one of the otheraforementioned constant region genes, e.g. γ (encoding about 330 aminoacids).

The term “immunogenicity” is used herein in its broadest sense toinclude the property of evoking an immune response within an organism.Immunogenicity typically depends partly upon the size of the substancein question, and partly upon how unlike host molecules it is. It isgenerally considered that highly conserved proteins tend to have ratherlow immunogenicity.

The term “immunoglobulin” is used herein to refer to a proteinconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes. The recognized immunoglobulin genes include the κ,λ, α, γ(IgG₁, IgG₂, IgG₃, IgG₄), δ, ε and μ constant region genes, aswell as the myriad immunoglobulin variable region genes. One form ofimmunoglobulin constitutes the basic structural unit of an antibody.This form is a tetramer and consists of two identical pairs ofimmunoglobulin chains, each pair having one light and one heavy chain.In each pair, the light and heavy chain variable regions are togetherresponsible for binding to an antigen, and the constant regions areresponsible for the antibody effector functions. In addition toantibodies, immunoglobulins may exist in a variety of other formsincluding, for example, Fv, Fab, Fab′ and (Fab′)₂.

Reference herein to “immuno-interactive” includes reference to anyinteraction, reaction, or other form of association between moleculesand in particular where one of the molecules is, or mimics, a componentof the immune system. An “immunointeractive molecule” includes anantibody, antibody fragment, synthetic antibody or a T-cell associatedbinding molecule (TABM).

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state.

A sample of biological fluid that is isolated from, or derived from, aparticular source of the host is described as being “obtained from”.

The invention also contemplates the use and generation of fragments ofmonoclonal antibodies produced by the method of the present inventionincluding, for example, Fv, Fab, Fab′ and F(ab′)₂ fragments. Suchfragments may be prepared by standard methods as for example describedby Coligan et al. (1991-1997, supra).

The present invention also contemplates synthetic or recombinantantigen-binding molecules with the same or similar specificity as themonoclonal antibodies of the invention. Antigen binding molecules ofthis type may comprise a synthetic stabilized Fv fragment. Exemplaryfragments of this type include single chain Fv fragments (sFv,frequently termed scFv) in which a peptide linker is used to bridge theN terminus or C terminus of a V_(H) domain with the C terminus orN-terminus, respectively, of a V_(L) domain. ScFv lack all constantparts of whole antibodies and are not able to activate complement.Suitable peptide linkers for joining the V_(H) and V_(L) domains arethose which allow the V_(H) and V_(L) domains to fold into a singlepolypeptide chain having an antigen binding site with a threedimensional structure similar to that of the antigen binding site of awhole antibody from which the Fv fragment is derived. Linkers having thedesired properties may be obtained by the method disclosed in U.S. Pat.No. 4,946,778. However, in some cases a linker is absent. ScFvs may beprepared, for example, in accordance with methods outlined in Krebber etal. (1997, supra). Alternatively, they may be prepared by methodsdescribed in U.S. Pat. No. 5,091,513, European Patent No 239,400 or thearticles by Winter and Milstein (Nature 349: 293, 1991) and Plückthun etal. (In Antibody engineering: A practical approach 203-252, 1996).

Alternatively, the synthetic stabilised Fv fragment comprises adisulphide stabilized Fv (dsFv) in which cysteine residues areintroduced into the V_(H) and V_(L) domains such that in the fullyfolded Fv molecule the two residues will form a disulphide bondtherebetween. Suitable methods of producing dsFv are described, forexample, in (Glockshuber et al., Biochem. 29: 1363-1367, 1990; Reiter etal., J. Biol. Chem. 269: 18327-18331, 1994; Reiter et al., Biochem. 33:5451-5459, 1994; Reiter et al., Cancer Res. 54: 2714-2718, 1994; Webberet al., Mol. Immunol. 32: 249-258, 1995).

Also contemplated as synthetic or recombinant antigen-binding moleculesare single variable region domains (termed dabs) as, for example,disclosed in (Ward et al., Nature 341: 544-546, 1989; Hamers-Castermanet al., Nature 363: 446-448, 1993; Davies & Riechmann, FEBS Lett. 339:285-290, 1994).

Alternatively, the synthetic or recombinant antigen-binding molecule maycomprise a “minibody”. In this regard, minibodies are small versions ofwhole antibodies, which encode in a single chain the essential elementsof a whole antibody. Suitably, the minibody is comprised of the V_(H)and V_(L) domains of a native antibody fused to the hinge region and CH3domain of the immunoglobulin molecule as, for example, disclosed in U.S.Pat. No. 5,837,821.

In an alternate embodiment, the synthetic or recombinant antigen bindingmolecule may comprise non-immunoglobulin derived, protein frameworks.For example, reference may be made to Ku & Schutz (Proc. Natl. Acad.Sci. USA 92: 6552-6556, 1995) which discloses a four-helix bundleprotein cytochrome b562 having two loops randomized to createcomplementarity determining regions (CDRs), which have been selected forantigen binding.

The synthetic or recombinant antigen-binding molecule may be multivalent(i.e. having more than one antigen binding site). Such multivalentmolecules may be specific for one or more antigens. Multivalentmolecules of this type may be prepared by dimerization of two antibodyfragments through a cysteinyl-containing peptide as, for exampledisclosed by (Adams et al., Cancer Res. 53: 4026-4034, 1993; Cumber etal., J. Immunol. 149: 120-126, 1992;). Alternatively, dimerization maybe facilitated by fusion of the antibody fragments to amphiphilichelices that naturally dimerize (Plünckthun, Biochem. 31: 1579-1584,1992) or by use of domains (such as leucine zippers jun and fos) thatpreferentially heterodimerize (Kostelny et al., J. Immunol. 148:1547-1553, 1992). In further embodiment, a multi-step process isemployed such as first administering a deimmunized antibody and then ananti-antibody with, for example, a reporter molecule.

The present invention further encompasses chemical analogues of aminoacids in the variant antibodies. The use of chemical analogues of aminoacids is useful inter alia to stabilize the molecules when administeredto a subject. The analogues of the amino acids contemplated hereininclude, but are not limited to, modifications of side chains,incorporation of unnatural amino acids and/or their derivatives duringpeptide, polypeptide or protein synthesis and the use of crosslinkersand other methods which impose conformational constraints on theproteinaceous molecule or their analogues.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. A list of unnaturalamino acid, contemplated herein is shown in Table 3.

TABLE 3 Non-conventional Non-conventional amino acid Code amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilize 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, peptides can beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

A monoclonal antibody obtained before deimmunization may be identifiedby any number of means including the steps of:

(a) coating a surface with antigen selected from cross-linked fibrinderivative or extract containing same or fibrinogen degradation product;

(b) contacting the antigen in step (a) with monoclonal antibody derivedfrom fibrin cross-linked derivative prepared as described above; and

(c) subjecting the complex formed in step (b) to a signal amplificationstep.

Suitably, in step (a), a well plate may be utilized in whichcross-linked fibrin derivatives such as D-dimer and/or fibrinogendegradation product (preferably obtained from a procedure whereinfibrinogen was suitably digested with thrombin to obtain fragment D,fragment E and optionally fragments X and Y) was applied to theindividual wells.

Subsequently, monoclonal antibody derived from a cross-linked fibrinderivative was then added to each well. An appropriate signalamplification step which may be applied is an EIA step wherein anappropriate enzyme conjugate may be coupled to the complex and substratesubsequently added. Alternatively, RIA, FIA, agglutination, adherence orchemiluminescence may be used as appropriate signal amplification steps.

The purpose of the screening assay procedure referred to above is toensure that the cells being tested are producing antibody specific tothe relevant cross-linked fibrin derivative, but not to fragment D.

There should be minimal reaction with fibrinogen or fibrinogendegradation products and a positive reaction with the derivative. Theterm “minimal” includes no reactivity but extends to basal levels suchas compared to an antibody-directed to fibrinogen per se. Consequently,a minimal reaction includes sub-optimal reactivity compared to afibrinogen-specific antibody.

The present invention also includes within its scope an assay to detectlinked fibrin derivatives including the steps of

(1) contacting a monoclonal antibody specific to cross-linked fibrinderivatives but not fragment D with a biological sample suspected ofcontaining an antigen derived from a cross-linked fibrin derivative orcomprising a cross-linked fibrin derivative per se; and

(2) subjecting the complex formed in step (1) to a signal amplificationstep.

In the above-mentioned assay, the cross-linked fibrin derivative issuitably D-dimer, D₂E or any other derivative of a high molecular weightnature as described above. The monoclonal antibody is prepared asdescribed previously which is relevant to the particular cross-linkedfibrin derivative being assayed.

The presence of the cross-linked fibrin derivative may be used as asuitable diagnostic aid for prethrombotic, thrombotic or otherconditions that involve the formation and lysis of fibrin.

The deimmunized monoclonal antibody of the present invention isparticularly useful for blood clot imaging as well as for targetingblood clots in order to bring the clot into contact with enzymes orother chemical agents capable of dissolving, wholly or partially, theclot.

With respect to clot imaging, a reporter molecule is attached to thedeimmunized monoclonal antibody or to an antibody having specificity forthe deimmunized antibody or a portion or conjugate thereon and this isthen introduced to a host, such as a human. By detecting the reportermolecule, blood clots can be visualized. One particularly useful form ofreporter molecule is a nuclear tag. Nuclear tags contemplated for use inthe present invention include but not limited to a bifunctional metalion chelate. The chelate may be attached to the antibody itself ormultiple chelates may be attached to the protein via dendrimers.Particularly preferred nuclear tags are ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga,⁷⁷Br, ⁹⁷Ru, ¹¹¹In, ¹²³I, ¹²⁴I, ¹³¹I and ¹⁸⁸Re. The most preferrednuclear tag is ^(99m)Tc. Preferably, the host is a human and, hence, itis necessary for the 3B6 murine monoclonal antibody to be deimmunized.

Alternative forms of immunoscintigraphy may be obtained using isotopessuch as a ⁶⁸Ga or ¹²⁴I or other PET isotopes. Such technology may bedescribed as “immuno-PET”. The technology has advantages over γ camerascintigraphy and may provide high resolution images of blood clotsespecially in areas of the body less amenable to conventional diagnosticmeans such as lungs or small clots in the calf or pelvis.

Accordingly, the present invention provides a conjugate moleculecomprising a deimmunized immunointeractive molecule such as adeimmunized antibody and one or both of an imaging tag or a therapeuticagent.

Preferred imaging tags are MRI-, ultrasound- and/or CT-type tags such asbut not limited to ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁹⁷Ru, ¹¹¹In,¹²³I, ¹²⁴I, ¹³¹I and ¹⁸⁸Re.

Preferred therapeutic tags include cytokines, anti-clotting agents,wound-repairing agents and anti-infection agents.

Another aspect of the present invention contemplates a method fordetecting a blood clot in a human patient, said method comprisingintroducing into said patient a deimmunized form of murine monoclonalantibody 3B6 or an antigen-binding fragment thereof labeled with areporter molecule allowing dissemination of the labeled antibodythroughout the circulatory system and then subjecting said patient toreporter molecule-detection means to identify the location of theantibody in a clot.

Preferably, the reporter molecule is a nuclear tag.

Preferably, the nuclear tag is ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁹⁷Ru, ¹¹¹In, ¹²³I, ¹²⁴I, ¹³¹I and ¹⁸⁸Re.

Preferably, the nuclear tag is ^(99m)Tc.

The present invention further contemplates the use of a deimmunizedmurine monoclonal antibody specific for D-dimer or other cross-linkedfibrin derivatives in the manufacture of clot imaging agent.

Preferably, the murine monoclonal antibody is 3B6 or a homologuethereof.

Preferably, the clot imaging tag is for use in humans.

The same antibody may also carry multiple tages such as multipleanti-coagulant agents and/or reporter molecules. Alternatively, or inaddition, multiple anti-antibodies may be administered each carrying adifferent tag.

The present clot targeting antibody may be used alone or in combinationwith other imaging protocols. One such protocol is planar imaging suchas but not limited to CT, MRI or ultrasound.

Accordingly, another aspect of the present invention contemplates amethod for detecting a blood clot in a human patient, said methodcomprising introducing into said patient a deimmunized form of murinemonoclonal antibody 3B6 or an antigen-binding fragment thereof labeledwith a reporter molecule allowing dissemination of the labeled antibodythroughout the circulatory system and then subjecting said patient toplanar clot imaging.

Preferably, the planar imaging is MRI or CT scanning. Ultrasound mayalso be used in the imaging process.

Accordingly, another aspect of the present invention contemplates amethod for detecting a blood clot in a human patient, said methodcomprising introducing into said patient a deimmunized form of murinemonoclonal antibody 3B6 or an antigen-binding fragment thereof labeledwith a reporter molecule allowing dissemination of the labeled antibodythroughout the circulatory system and then subjecting said patient to acomputer assisted tomographic nuclear medicine scan to visualize theclot.

Preferably, the reporter molecule is a nuclear tag.

Preferably, the nuclear tag is ^(99m)TC, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁹⁷Ru, ¹¹¹In, ¹²³I, ¹²⁴I, ¹³¹I and ¹⁸⁸Re.

Preferably, the nuclear tag is ^(99m)Tc.

The clot imaging agents of the present invention are also useful astherapeutic agents. In particular, the clot targeting agents are fused,bound or otherwise associated with a clot dissolution or clot growthprevention agent such as an anticoagulant molecule.

Accordingly, another aspect of the present invention contemplates amethod for facilitating the dissolution or removal of a blood clot in ahuman, said method comprising administering to said human a clotdissolution or clot growth prevention-effective amount of a variantmurine-derived monoclonal antibody having specificity for human-derivedD-dimer and other cross-linked fibrin derivatives and non-reactivitywith fibrinogen or fibrinogen degradation products inclusive offragments D and E wherein said variant murine-derived monoclonalantibody is substantially non-immunogenic in a human wherein saidmonoclonal antibody further comprises a clot dissolution or clot growthprevention agent fused, bound or otherwise associated thereto.

Yet another aspect of the present invention is directed to the use of avariant murine-derived monoclonal antibody having specificity forhuman-derived D-dimer and other cross-linked fibrin derivatives andnon-reactivity with fibrinogen or fibrinogen degradation productsinclusive of fragments D and E wherein said variant murine-derivedmonoclonal antibody is substantially non-immunogenic in a human and saidantibody further comprising a clot dissolution or clot growth preventionagent fused, bound or otherwise attached thereto in the manufacture of amedicament for the dissolution of a blot clot in a human.

In an alternative embodiment, multiple deimmunized antibodies may beused. In one example, a deimmunized 3B6 antibody is administered aloneand then deimmunized anti-immunglobulin antibodies each carrying anagent such as a diagnostic or therapeutic agent which will target aclot-3B6 complex. Yet another alternative is to engineer antibodies withmultiple (e.g. bi-) specificities. In this case, one specificity may beto the clot and another to the site of the clot (e.g. to a cellreceptor). This may also be accomplished using multiple antibodies.

The present invention further contemplates compositions comprising theclot targeting agents of the present invention and one or morepharmaceutically acceptable carriers and/or diluents.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions as well as lyophilized forms of antibody preparationstogether with stabilizing agents such as sugar, proteins or othercompounds or molecules which facilitate the radiolabeling process. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dilution mediumcomprising, for example, water, ethanol, polyol (for example, glycerol,propylene glycol and liquid polyethylene glycol, and the like), suitablemixtures thereof and vegetable oils. The proper fluidity can bemaintained, for example, by the use of superfactants. The preventions ofthe action of microorganisms can be brought about by variousanti-bacterial and anti-fungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminium monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with theactive ingredient and optionally other active ingredients as required,followed by filtered sterilization or other appropriate means ofsterilization.

Pharmaceutically acceptable carriers and/or diluents include any and allsolvents, dispersion media, coatings, anti-bacterial and anti-fungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art and except insofar as any conventional media or agent isincompatible with the active ingredient, their use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The clot targeting agents of the present invention are useful for thediagnosis and/or treatment of thrombin-associated conditions such asDVT, PE and DIC.

Yet another aspect of the present invention contemplates a method fortreating a subject with cancer associated with fibrin. In thisembodiment, antibodies to the Dimer epitope may be used to delivercytotoxic agents such as an isotope that emits β or γ emission orcombinations thereof. Such isotopes include but are not limited to ¹³¹I,yttrium-90, rhenium-186, rhenium-188, lutetium-117 and copper-67. Fibrinassociated with a cancer includes a fibrin encapsulated tumor.

The deimmunized immunointeractive molecules of the present inventionare, therefore, carriers for any clot binding agents or clot dissolvingagents or for any agents which have useful diagnostic or therapeuticproperties. The deimmunized immunointeractive molecules of the presentinvention are also useful for determining the kinetics of clotdissolution, dissipation and/or disappearance. One this information isavailable, clot dissolving or imaging agents can very quickly beadministered.

The present invention is further described by the following non-limitingExamples.

Example 1 Cell Fusion and Selection of Hybrids

Spleens were removed aseptically from 2 immunized mice killed bycervical dislocation three days after an injection of D-dimer.Previously, the mice had been immunized with three injections of fibrinlysate digested with proteolytic enzymes thrombin and plasmin asreported in the aforementioned Graeff and Hafter reference. Two spleenswere placed in a 60 mm Petri dish (Falcon, 3001, Oxnard, Calif.)containing 5 ml complete medium (85% RPMI 1640, 15% w/v fetal calfserum, 100 I.U./ml penicillin, 100 μg/ml streptomycin and 2×10⁻³ Mglutamine; Gibco, Grand Island, N.Y.). A cell suspension was prepared bydecapsulating the spleen with 2×18 gauge needles attached to 3 mldisposable syringes with the last cm of the tip bent through an angle of60°. The cell suspension was then aspirated into a 10 ml syringe fittedwith a 22 gauge needle and ejected with moderate pressure. Thisoperation was performed twice before filtering the cells into a Falcon2001 tube through a fine mesh stainless steel screen to remove largercell clumps and debris.

The cell suspension was allowed to stand for 5 minutes at roomtemperature to allow smaller clumps and membrane fragments to settlebefore transferring the cell suspension to a fresh Falcon 2001 tube. Thecells were centrifuged at 350 G for 5 minutes at room temperature andthe supernatant was decanted from the first cell pellet to a fresh tubeand spun at 700 G for five minutes to give a second cell pellet and thetwo pellets were pooled and resuspended in 5 ml complete medium. Thespleen white blood cells (SWBC) were then counted and their viabilityestimated by Turks and Trypan blue stains, respectively, and 100×10⁶viable SWBC were placed in separate Falcon 2001 tubes in a total volumeof 5 ml complete medium. The NS-1 myeloma cells to be used for fusion,were washed once by centrifugation at 380 G for 15 minutes at roomtemperature and adjusted to 5×10⁶ viable cells/ml in complete medium.

Twenty-five×10⁶ NS-1 and 100×10⁵ immune SWBC were mixed and spun at 350G for 5 minutes at room temperature. The supernatant was decanted, theremaining medium was carefully removed with a Pasteur pipette and 2 mlof a 42% w/v solution of polyethylene glycol (PEG, MW1540) (BakerChemical Co., New Jersey). In RPMI 1640 containing 15% v/v dimethylsulfoxide (DMSO) at 37° C. was added with a 5 ml glass disposablepipette (Corning Glass, Corning, N.Y.) and the cells were resuspendedwith the same 5 ml pipette for 30 seconds with the aid of an electricpipetter (Pipet-aid Drummond Scientific Co., Broomall, Pa.). ThePEG-cell suspension was allowed to stand for a further 30 seconds atroom temperature before adding 5 ml complete medium, dropwise, with aPasteur pipette, over a period of 90 seconds with constant flicking ofthe tube, sufficient to ensure complete mixing with the viscous PEGsolution. A further 5 ml complete medium was immediately added and mixedby inversion and the cell suspension was allowed to stand for a further150 seconds at room temperature before centrifugation at 350 G for 5minutes at room temperature. The supernatant was decanted and the cellpellet was gently resuspended in 5 ml complete medium using a 5 mlpipette with the electric pipetter; extreme care was taken not to breakup all cell clumps. Using a Tridak stepper (Bellco Glass Inc., Vineland,N.J.), 0.05 ml of the cell suspension was added to each well of 4 Costar24 well plates (Costar 3524, Cambridge, Mass.) containing 1×10⁶ normalBALB/c mouse SWBC as feeder cells in 1 ml complete medium containing10⁻⁴ M Hypoxanthine (Sigma), 4×10⁻⁷ M Aminopterin (Sigma), 1.6×10⁻⁵ MThymidine (Sigma) and 4×10⁻⁵ M 2-Mercaptoethanol (HAT medium),hereinafter referred to as 1° fusion plates.

The 1° fusion plates were then placed in a humidified 5% CO₂ 95% airatmosphere at 37° C. The cells were first fed either on days 5 or 7 andthereafter when necessary, with 0.5 ml fresh HAT medium. Generally, onday 10, 0.5 ml of the medium was removed for the screening assay fromeach well showing hybridoma growth and 0.5 ml fresh HAT medium wasreplaced. A number of the strongest growth wells were chosen formaintenance on the basis of the screening assay. The chosen wells wereallowed to grow to confluency in the original original well (1° well),then each was split in half and transferred to a fresh well (2° well) ofa 24 well Costar plate (2° plate). The wells were checked daily andexpanded to a second, third or fourth well of the 2° Costar plate whennecessary. From days 14-28, cells were fed with HT medium. When therewas strong growth in at least two wells of the 2° plate, supernatantfrom one well of each clonotype was chosen for rescreening and a numberof specific antibody producing clonotypes were chosen from the resultsof the second screening assay to produce monoclonal antibody secretingcell lines by limiting dilution.

Example 2 Cloning of hybridomas

One 2° well of each chosen clonotype was resuspended and the number ofviable cells per well was estimated by Trypan blue exclusion.Immediately before plating each clonotype, the relevant series ofdilutions were made in HT medium or complete medium (if the cells wereolder than 28 days post fusion) to give a frequency of 0.5 cells/0.05ml. This volume was then added with a Tridak stepper to each well of a96 well flat bottomed tissue culture plate (Flow Laboratories,Mississauga, Ontario, Canada) (LD plate) containing 1×10⁵ normal mousespleen feeder cells in 0.1 ml HT or complete medium. the LD plates werethen placed in a 37° C. humidified 5% CO², 95% air atmosphere andscreened for clonal growth 7-10 days later. From each positive growthwell, 0.1 ml supernatant was removed for screening and these wells werefed for the first time with 0.1-0.15 ml HT or complete medium. On thebasis of the LD screening assay, a minimum of two of the “better”specific antibody-producing clones were finally selected for expansionto mass culture.

Alternatively, if it was desired to obtain a large amount of Mab, femaleBALB/c mice were given an intraperitoneal injection of 0, 5 ml 2, 5, 10,14, tetramethylpentadecane (Pristane, Aldrich Chemical Corp., Milwaukee,Wisonsin) 14 days prior to the injection of 2×10⁶ viable hybridoma cellsand ascites fluids were collected from the mice 12-14 days afterinjection of the cells. The ascitic fluid was clarified bycentrifugation and MAb recovered by precipitation with 45% ammoniumsulphate and stored at either 4° C. or −70° C. in phosphate bufferedsaline (PBS) containing 0.01% sodium azide.

Example 3 Monoclonal Antibody Screening Assay

The wells of a 96 well U bottomed microtest plate (Disposable ProductsPty. Ltd., Adelaide, South Australia) were coated by adding 50 μl ofeither D-dimer (5 μg/ml) or Fibrinogen degradation products (5 μg/ml inPBS for one hour at room temperature (25° C.). Excess antigen wasremoved by inverting and tapping the plate and the plate was then washedthree times with PBS containing 0.05% w/v Tween 20 (Sigma ChemicalCorp., St Louis, Mo.). Clones secreting MAb to D-dimer or Fibrinogendegradation products were then detected by adding 50 μl of tissueculture supernatant to each well and incubating for one hour at roomtemperature. Unbound MAb was removed by inversion and tapping and theplate was washed three times with PBS/Tween. One hundred μl of a 1/1000dilution of peroxidase conjugated rabbit anti-mouse immunoglobulin(Dakopatts, Copenhagen, Denmark) in PBS/Tween was added and allowed toincubate a further one hour at room temperature. The plate was againinverted and washed three times with PBS/Tween and 100 μl of activatedsubstrate (immediately before use, 10 μl of 3% solution ofhydrogen-peroxide was added to 10 ml of a substrate solution containing50 mM citrate, 2.5 mM of O-tolidine dihydrochloride (O-tolidine, SigmaChemical Co., recrystallized from dilute HCl) 0.025 mM EDTA pH 4.5) wasadded to each well. The colour reaction was stopped after 10 minutes bythe addition of 50 μl of 3M HCl which caused a colour change from blueto yellow and the absorbance was recorded at 450 nm on a Titertekmultiskan.

Example 4 Identification of 3B6 Variable Region Sequences

The murine hybridoma 3B6 was propagated in RPMI 1640 medium supplementedwith 15% w/v fetal calf serum. Total RNA was prepared from 10⁷ hybridomacells. V_(H) and V^(K) cDNA was prepared using reverse transcriptase andmouse κ constant region and mouse IgG constant region primers. The firststrand cDNAs were amplified by PCR using a variety of mouse signalsequence primers (6 sets for V_(H) and 7 sets for V_(K)). The amplifiedDNAs were gel-purified and cloned into the vector pGem® T Easy(Promega). The V_(H) and V_(K) clones obtained were screened for insertsof the expected size by PCR and the DNA sequence of selected clonesdetermined by the dideoxy chain termination method.

Productive V_(H) and V_(K) genes were identified by sequence analysis.The location of the complementarity determining regions (CDRs) wasdetermined with reference to other antibody sequences (43). The 3B6V_(H) can be assigned to mouse heavy chains sub-group IA. The 3B6 V_(K)can be assigned to mouse K chains sub-group I.

Example 5 Analysis of 3B6 Variable (v) Region Sequences with Potential TCell Epitopes

3B6 V_(H) and V_(K) sequences were analyzed for the presence ofpotential T cell epitopes using procedures described previously (Carr etal., International Patent Publication No. WO 98/52976). The peptidesidentified as potential T cell epitopes (MHC class II binding peptides)were modified in silico and the modified sequence re-analyzed to ensureloss of potential MHC class II binding and verify that further MHC classII binding motifs had not been generated in the surrounding sequence.Alternatively, the sequence was modified to convert the MHC class IIbinding motif to one found in the human germ line. Single, generallyconservative, amino acid substitutions were tested and substitutionsmade with due regard to overall antibody structure. A number of variantsequences are compiled for the V_(H) and V_(K), each containingdifferent numbers of substitutions.

Example 6 Designer Variant 3B6 Variable (v) Regions Sequences withReduced Numbers of Potential T Cell Epitopes

The heavy and light (v) regions designed according the scheme of Example5 were constructed in vitro by the method of overlapping PCRrecombination described (Daugherty et al., Nucleic Acids Research 19:2471-2476, 1991). The cloned murine V_(H) and V_(K) genes were used astemplates for mutagenesis of the framework regions to the requiredhumanized sequences. Sets of mutagenic primer pairs were synthesizedencompassing the regions to be altered. Adjacent primers included 15 bpof homologous sequence. A first round of PCR using these primersproduced 5 to 8 overlapping DNA fragments encompassing the designed (y)region gene. The vectors V_(H)-PCR1 and V_(K)-PCR1 (Orlandi et al.,Proc. Natl. Acad. Sci. USA 86: 3833-3837, 1989) were used as templatesto introduce 5′ flanking sequence including the leader signal peptide,leader intron and the murine immunoglobulin promoter, and 3′ flankingsequence including the splice site and intron sequences, in anadditional two overlapping fragments. The DNA fragments produced werecombined in a second round of PCR using outer flanking primers to obtainPCR products of the required full length. These PCR products were clonedinto the vector pUC19 for DNA sequence determination. Clones wereselected that contained the expected sequence alterations and the entireDNA sequence was confirmed to be correct for each desired V_(H) andV_(K). The heavy and light chain genes were transferred to theexpression vectors pSVgpt and pSVhyg with human IgG1 or K constantregions as described (Tempest et al., Biotechnology 9: 266-271, 1991).The vectors V_(H)-PCR1 and V_(K)-PCR1 (Orlandi et al., 1989, supra) wereused as templates to introduce 5′ flanking sequence including the leadersignal peptide, leader intron and the murine immunoglobulin promoter,and 3′ flanking sequence including the splice site and intron sequences.

Example 7 Expression and Purification of Variant 3B6 Antibodies

The variant 3B6 heavy and light chain expression vectors wereco-transfected in different combinations by electroporation into NS0, anon-immunoglobulin producing mouse myeloma, obtained from the EuropeanCollection of Animal Cell Cultures, Porton, U.K. (ECACC No 85110505).Colonies expressing the gpt gene were selected in Dulbecco's ModifiedEagle's Medium (DMEM) supplemented with 0.8 μg/ml mycophenolic acid and250 μg/ml xanthine. Production of human antibody by transfected cellclones was measured by ELISA for human IgG (48). Cell lines secretingantibody were selected and expanded. Variant 3B6 antibodies werepurified using Prosep®-A (Bioprocessing Ltd, Conset, U.K.).

Example 8 Functional Testing of Variant 3B6 Antibodies

Variant antibodies were tested for D-dimer binding using ELISA basedassays broadly as described in Example 3. Binding specificity wasconfirmed using the human fibrinogen binding assay. In a preferredembodiment, however, the D-dimer was used in solution phase. In thisassay, the 3B6 antibodies were coated on the ELISA plate at 0.5 μg/well,to capture D-dimer in solution. D-dimer was applied at 10 μg/ml (500ng/well) and doubling dilutions. The revealing antibody was HRPOconjugated mouse monoclonal anti-D (Dimertest EIA Tag; Agen) and theresults were developed by OPD substrate and read a 492 nm. Thedeimmunized 3B6 antibodies are compared to the murine and chimeric 3B6antibodies and the previous lead deimmunized antibody 3B6 DWH1/DIK1. Theresults are shown in FIGS. 4A, 4B and 4C. The use of solution phaseD-dimer proved better than solid phase D-dimer in the selection ofclones and is a preferred aspect of the present invention.

Example 9 Thromboviewing Using 3B6-99mTc

The 3B6 monoclonal antibody from mice and deimmunized form for humans isrepresented in FIG. 1A and exhibits specificity for fibrin which is amajor part of blood clots (FIG. 1B). A clot imaging concept is developedby labelling the 3B6 monoclonal antibody with a nuclear tag, in thiscase, ^(99m)Tc (FIG. 2). Administration of the labeled 3B6 deimmunizedmonoclonal antibody in humans (FIG. 3A). Visualization of clots in thecirculatory system such as blood clots in the anterior thighs (FIG. 3B)occurs by binding of the monoclonal antibody to fibrin resulting inconcentration of radiation at the clot site.

Example 10 Thromboviewing Using 3B6-^(99m)Tc

Pulmonary emboli (0.1-0.5 g) were created in anesthetized dogs byembolization of pre-formed thrombi made by infusion of thrombin andhuman fibrinogen through balloon catheters placed in the femoral veins.Purified Fab′ fragments (0.35 mg) of a chimeric (human/murine)derivative of a fibrin-specific monoclonal antibody were labeled with a15 mCi of ^(99m)Tc. One hour after embolization, the radiolabeledantibody preparation was injected through a peripheral intravenouscatheter. Eight hours after antibody injection, imaging scans wereperformed to visualize the emboli.

^(99m)Tc labeled antibody fragments cleared from the circulation with at_(1/2) of one hour for both subjects. In subject 1, two small emboli inthe right lower lobe (combined mass, 0.187 g) were visible. Theclot/blood radioactivity ratio was 38:1. In subject 2, one embolus inthe right lower lobe (mass, 0.449 g) was visible. Clot/bloodradioactivity ratio was 27:2. A small embolus (0.091 g) was discoveredin the right ventricle of subject 1. The clot/blood radioactivity ratiowas 45:1. No adverse effects were noted from either antibodyadministration or scanning methodology.

Infusion of radiolabeled anti-fibrin antibody fragments followed byimaging produces images of emboli, even relatively small emboli in theperiphery of the lung. The images are reliable and require minimaltraining to interpret. The technique can be used to image deep veinthrombi in the same setting. This agent is well tolerated by thesubjects. There is no need for breath-holding or cardiac gating. It usesno nephrotoxic intravenous contrast dye. The radiation dose is similarto the dose used for ventilation/perfusion scans. This: technology maysimplify and clarify the diagnosis of PE and DVT, using technologyavailable in most medical centres.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

All publications, patents, patent applications and provisionalapplications referred to herein are incorporated herein by reference intheir entirety.

BIBLIOGRAPHY

-   Adams et al., Cancer Res. 53: 4026-4034, 1993.-   Altschul et al., Nucl. Acids Res. 25:3389-3402. 1997.-   Ausubel et al., “Current Protocols in Molecular Biology” John Wiley    & Sons Inc, 1994-1998, Chapter 15.-   Budzynski et al., Blood 54(4), 1979.-   Carr et al. (International Patent Publication No. WO 98/52976).-   Chothia et al., J. Mol. Biol. 196: 901, 1987.-   Chothia et al., J. Mol. Biol. 227: 799, 1992.-   Chou et al. (U.S. Pat. No. 6,056,957).-   Coligan et al., Current Protocols in Immunology, John Wiley & Sons,    Inc., 1991-1997.-   Cumber et al., J. Immunol. 149: 120-126, 1992.-   Daugherty et al., Nucleic Acids Research 19: 2471-2476, 1991.-   Davies & Riechmann, FEBS Lett. 339: 285-290, 1994.-   European Patent Publication No. 0 239 400.-   Gefter et al., Somatic Cell Genet. 3: 231-236, 1977.-   Glockshuber et al., Biochem. 29: 1363-1367, 1990.-   Graeff and Halfer, “Detection and Relevance of Cross-linked Fibrin    Derivatives in Blood”, Seminars in Thrombosis and Hemostatis 8(1),    1982.-   Hamers-Casterman et al., Nature 363: 446-448, 1993.-   Jones et al., Nature 321: 522-525, 1986.-   Kabat et al., “Sequences of Proteins of Immunological Interest”,    U.S. Department of Health and Human Services, 1983.-   Kennet et al. (eds) Monoclonal Antibodies and Hybridomas: A New    Dimension in Biological Analyses, pp. 376-384, Plenum Press, New    York, 1980.-   Kohler and Milstein, Eur. J. Immunol. 6(7): 511-519, 1976.-   Kohler and Milstein, Nature 256: 495-499, 1975.-   Kostelny et al., J. Immunol. 148: 1547-1553, 1992.-   Kozbor et al., Methods in Enzymology 121: 140, 1986.-   Krebber et al., J. Immunol. Methods 201(1): 35-55, 1997.-   Ku & Schutz, Proc. Natl. Acad. Sci. USA 92: 6552-6556, 1995.-   Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987.-   Morgan et al. U.S. Pat. No. 6,180,377).-   Orlandi et al., Proc. Natl. Acad. Sci. USA 86: 3833-3837, 1989.-   Plückthun et al., In Antibody engineering: A practical approach    203-252, 1996.-   Plückthun, Biochem. 31: 1579-1584, 1992.-   Queen et al. (U.S. Pat. No. 6,180,370).-   Reiter et al., Biochem. 33: 5451-5459, 1994.-   Reiter et al., Cancer Res. 54: 2714-2718, 1994.-   Reiter et al., J. Biol. Chem. 269: 18327-18331, 1994.-   Riechmann et al., Nature 332: 323-327, 1988.-   Shulman et al., Nature 276: 269-270, 1978;-   Tempest et al., Biotechnology 9: 266-271, 1991.-   Toyama et al., “Monoclonal Antibody, Experiment Manual”, published    by Kodansha Scientific, 1987.-   Trowbridge, J. Exp. Med. 148(1): 313-323, 1978.-   Verhoeyen et al., Science 239: 1534-1536, 1989.-   Volk et al., J. Virol. 42(1): 220-227, 1982.-   Ward et al., Nature 341: 544-546, 1989.-   Webber et al., Mol. Immunol. 32: 249-258, 1995.-   Willner et al., Biochemistry 21: 2687-2692, 1982.-   Winter and Milstein, Nature 349: 293, 1991,

1. An isolated antibody specific for an epitope on human D-dimer whichrecognizes cross-linked fibrin but not fibrinogen, wherein said antibodyis selected from the group consisting of: (i) an antibody that comprisesa binding site for said D-dimer epitope, wherein one or more amino acidresidues in a T-cell epitope present in the variable (v)-domain of saidantibody are mutated to eliminate or reduce the ability of peptidefragments of said v-domain to associate with MHC class II molecules; and(ii) an antibody that comprises complementary determining regions (CDRs)that define a binding site for said D-dimer epitope, interspersedbetween framework regions that comprise a human antibody light and heavychain variable region.
 2. The isolated antibody of claim 1 wherein saidantibody is specific for an epitope recognized by anti-fibrin murinemonoclonal antibody 3B6.
 3. The isolated antibody of claim 1, whereinthe antibody comprises an H-chain selected from the group consisting ofHv5 (3B6DIVHv5; SEQ ID NO:1), Hv6 (3B6DIVHv6; SEQ ID NO:2) and Hv7(3B6DIVHv7; SEQ ID NO:3).
 4. The isolated antibody of claim 1, whereinthe antibody comprises an L-chain selected from the group consisting ofKv1 (3B6DIVKv1; SEQ ID NO:4), Kv4 (3B6DIVKv4; SEQ ID NO:5) and Kv7(3B6DIVKv7; SEQ ID NO:6).
 5. The isolated antibody of claim 1, whereinsaid antibody comprises a combination of an H-chain and an L-chainselected from the group consisting of VHv5/VKv1, VHv6/VKv1, VHv7/VKv1,VHv5/VKv7, VHv6/VKv7, VHv6/VKv4, VHv7/VKv4, VHv7/VKv7 and VHv5/VKv4. 6.The isolated antibody of claim 1, wherein said antibody comprises acombination of an H and an L chain v-domain comprising a combination ofamino acid sequences selected from the group consisting of SEQ IDNO:1/SEQ ID NO:4, SEQ ID NO:2/SEQ ID NO:4, SEQ ID NO:3/SEQ ID NO:4, SEQID NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQ ID No:6, SEQ ID NO:2/SEQ ID NO:5,SEQ ID NO:3/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:6, and SEQ ID NO:1/SEQ IDNO:5 or combinations of amino acid sequences having at least 70%sequence identity to the contiguous sequence of one or both amino acidsequences in each of the above listed pairs.
 7. The antibody of claim 6,wherein said antibody is an Fab′ fragment.
 8. The antibody of claim 1,wherein said antibody is selected by binding to D-dimer that is notpassively bound to a substrate.
 9. The antibody of claim 1, wherein saidantibody is selected by binding to D-dimer in solution.
 10. Adeimmunized antibody specific for an epitope on human D-dimer whichrecognizes cross-linked fibrin but not fibrinogen, wherein said antibodycomprises (a) an H-chain selected from the group consisting of Hv5(3B6DIVHv5; SEQ ID NO:1), Hv6 (3B6DIVHv6; SEQ ID NO:2) and Hv7(3B6DIVHv7; SEQ ID NO:3), and (b) an L-chain selected from the groupconsisting of Kv1 (3B6DIVKv1; SEQ ID NO:4), Kv4 (3B6DIVKv4; SEQ ID NO:5)and Kv7 (3B6DIVKv7; SEQ ID NO:6), and further wherein said antibody isselected by binding to D-dimer in solution.
 11. The deimmunized antibodyof claim 10, wherein said antibody comprises a combination of an H andan L chain v-domain comprising a combination of amino acid sequencesencoded by a combination of nucleotide sequences selected from the groupconsisting of SEQ ID NO:7/SEQ ID NO:10, SEQ ID NO:8/SEQ ID NO:10, SEQ IDNO:9/SEQ ID NO:10, SEQ ID NO:7/SEQ ID NO:12, SEQ ID NO:8/SEQ ID NO:12,SEQ ID NO:8/SEQ ID NO:11, SEQ ID NO:9/SEQ ID NO:11, SEQ ID NO:9/SEQ IDNO:12, and SEQ ID NO:7/SEQ ID NO:11.
 12. The deimmunized antibody ofclaim 10, wherein said antibody comprises a combination of an H and an Lchain v-domain comprising a combination of amino acid sequences selectedfrom the group consisting of SEQ ID NO:1/SEQ ID NO:4, SEQ ID NO:2/SEQ IDNO:4, SEQ ID NO:3/SEQ ID NO:4, SEQ ID NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQID No:6, SEQ ID NO:2/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:5, SEQ IDNO:3/SEQ ID NO:6, and SEQ ID NO:1/SEQ ID NO:5.
 13. The deimmunizedantibody of claim 10, wherein said antibody is an Fab′ fragment.
 14. Anisolated antibody wherein said antibody comprises human constantregions, and variable regions specific for an epitope recognized byanti-fibrin murine monoclonal antibody 3B6.
 15. The antibody of claim14, wherein said antibody comprises a combination of heavy and lightchain v-regions from VHv5 and VKv1.
 16. The antibody of claim 15,wherein said antibody comprises a heavy chain v-region comprising aminoacid residues 26-36, 51-71 and 99-107 of SEQ ID NO:1.
 17. The antibodyof claim 15, wherein said antibody comprises a light chain v-regioncomprising amino acid residues 24-34, 50-56 and 89-97 of SEQ ID NO:4.18. The antibody of claim 15, wherein said antibody comprises a heavychain v-region comprising amino acid residues 26-36, 51-71 and 99-107 ofSEQ ID NO:1 and a light chain v-region comprising amino acid residues24-34, 50-56 and 89-97 of SEQ ID NO:4.
 19. An isolated antibody thatcomprises human constant regions specific for an epitope recognized byanti-fibrin murine monoclonal antibody 3B6, and variable regionsspecific for an epitope recognized by anti-fibrin murine monoclonalantibody 3B6.
 20. A deimmunized antibody specific for an epitope onhuman D-dimer which recognizes cross-linked fibrin but not fibrinogen,wherein said antibody comprises a combination of an H and an L chainv-domain comprising a combination of amino acid sequences selected fromthe group consisting of SEQ ID NO:1/SEQ ID NO:4, SEQ ID NO:2/SEQ IDNO:4, SEQ ID NO:3/SEQ ID NO:4, SEQ ID NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQID No:6, SEQ ID NO:2/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:5, SEQ IDNO:3/SEQ ID NO:6, and SEQ ID NO:1/SEQ ID NO:5, or combinations of aminoacid sequences having a conservative amino acid substitution in one orboth amino acid sequences in each of the above listed pairs.
 21. Aconjugate comprising an imaging tag and an antibody specific for anepitope on human D-dimer which recognizes cross-linked fibrin but notfibrinogen wherein said antibody is selected from the group consistingof: (i) an antibody that comprises a binding site for said D-dimerepitope, wherein one or more amino acid residues in a T-cell epitopepresent in the v-domain of said antibody are mutated to eliminate orreduce the ability of peptide fragments of said v-domain to associatewith MHC class II molecules; and (ii) an antibody that comprisescomplementary determining regions (CDRs) that define a binding site forsaid D-dimer epitope interspersed between framework regions thatcomprise a human antibody light and heavy chain variable region
 22. Theconjugate of claim 21 wherein said antibody is specific for an epitoperecognized by anti-fibrin murine monoclonal antibody 3B6.
 23. Theconjugate of claim 21, wherein said antibody comprises an H-chainselected from the group consisting of Hv5 (3B6DIVHv5; SEQ ID NO:1), Hv6(3B6DIVHv6; SEQ ID NO:2), and Hv7 (3B6DIVHv7; SEQ ID NO:3).
 24. Theconjugate of claim 21, wherein said antibody comprises an L-chainselected from the group consisting of Kv1 (3B6DIVKv1; SEQ ID NO:4), Kv4(3B6DIVKv4; SEQ ID NO:5), and Kv7 (3B6DIVKv7; SEQ ID NO:6).
 25. Theconjugate of claim 21, wherein said antibody comprises a combination ofan H-chain and an L-chain selected from the group consisting ofVHv5/VKv1, VHv6/VKv1, VHv7/VKv1, VHv5/VKv7, VHv6/VKv7, VHv6/VKv4,VHv7/VKv4, VHv7/VKv7 and VHv5/VKv4.
 26. The conjugate of claim 21,wherein said antibody comprises a combination of an H and an L chainv-domain comprising a combination of amino acid sequences selected fromthe group consisting of SEQ ID NO:1/SEQ ID NO:4, SEQ ID NO:2/SEQ IDNO:4, SEQ ID NO:3/SEQ ID NO:4, SEQ ID NO:1/SEQ ID NO:6, SEQ ID NO:2/SEQID No:6, SEQ ID NO:2/SEQ ID NO:5, SEQ ID NO:3/SEQ ID NO:5, SEQ IDNO:3/SEQ ID NO:6, and SEQ ID NO:1/SEQ ID NO:5 or combinations of aminoacid sequences having at least 70% sequence identity to the contiguoussequence of one or both amino acid sequences in each of the above listedpairs.
 27. The conjugate of claim 21 wherein said imaging tag is anx-ray, MRI, ultrasound-, CT-, or nuclear medicine-type tag.
 28. Theconjugate of claim 27 wherein the imaging tag is selected from the groupconsisting of ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁹⁷Ru, ¹¹¹In, ¹²³I,¹²⁴I, ¹³¹I and ¹⁸⁸Re.
 29. The conjugate of claim 28 wherein the imagingtag is ^(99m)Tc.
 30. The conjugate of claim 27, wherein the imaging tagis a gamma scintigraphy or PET-type tag.
 31. A conjugate comprising adeimmunized antibody according to claim 10, and an imaging tag selectedfrom the group consisting of ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁹⁷Ru, ¹¹¹In, ¹²³I, ¹²⁴I, ¹³¹I and ¹⁸⁸Re.
 32. A conjugate comprising adeimmunized antibody according to claim 11, and an imaging tag selectedfrom the group consisting of ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁹⁷Ru, ¹¹¹In, ¹²³I, ¹²⁴I, ¹³¹I and ¹⁸⁸Re.
 33. A conjugate comprising adeimmunized antibody according to claim 12, and an imaging tag selectedfrom the group consisting of ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁹⁷Ru, ¹¹¹In, ¹²³I, ¹²⁴I, ¹³¹I and ¹⁸⁸Re.
 34. A conjugate comprising adeimmunized antibody according to claim 13, and an imaging tag selectedfrom the group consisting of ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br,⁹⁷Ru, ¹¹¹In, ¹²³I, ¹²⁴I, ¹³¹I and ¹⁸⁸Re.
 35. A conjugate comprising anantibody according to claim 14, and an imaging tag selected from thegroup consisting of ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁹⁷Ru, ¹¹¹In,¹²³I, ¹²⁴I, ¹³¹I and ¹⁸⁸Re.
 36. A conjugate comprising an antibodyaccording to claim 19, and an imaging tag selected from the groupconsisting of ^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁹⁷Ru, ¹¹¹In, ¹²³I,¹²⁴I, ¹³¹I and ¹⁸⁸Re.
 37. A conjugate comprising an antibody accordingto claim 20, and an imaging tag selected from the group consisting of^(99m)Tc, ¹⁸F, ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷Br, ⁹⁷Ru, ¹¹¹In, ¹²³I, ¹²⁴I, ¹³¹I and¹⁸⁸Re.
 38. The conjugate of claim 31 wherein the imaging tag is^(99m)Tc.
 39. The conjugate of claim 32 wherein the imaging tag is^(99m)Tc.
 40. The conjugate of claim 33 wherein the imaging tag is^(99m)Tc.
 41. The conjugate of claim 34 wherein the imaging tag is^(99m)Tc.
 42. The conjugate of claim 35 wherein the imaging tag is^(99m)Tc.
 43. The conjugate of claim 36 wherein the imaging tag is^(99m)Tc.
 44. The conjugate of claim 37 wherein the imaging tag is^(99m)Tc.
 45. A method for detecting a blood clot in a human patient,said method comprising introducing into said patient a conjugateaccording to claim 21 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.
 46. A method for detecting a blood clot ina human patient, said method comprising introducing into said patient aconjugate according to claim 25 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.
 47. A method for detecting a blood clot ina human patient, said method comprising introducing into said patient aconjugate according to claim 26 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.
 48. A method for detecting a blood clot ina human patient, said method comprising introducing into said patient aconjugate according to claim 31 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.
 49. A method for detecting a blood clot ina human patient, said method comprising introducing into said patient aconjugate according to claim 33 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.
 50. A method for detecting a blood clot ina human patient, said method comprising introducing into said patient aconjugate according to claim 34 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.
 51. A method for detecting a blood clot ina human patient, said method comprising introducing into said patient aconjugate according to claim 38 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.
 52. A method for detecting a blood clot ina human patient, said method comprising introducing into said patient aconjugate according to claim 40 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.
 53. A method for detecting a blood clot ina human patient, said method comprising introducing into said patient aconjugate according to claim 41 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.
 54. A method for detecting a blood clot ina human patient, said method comprising introducing into said patient aconjugate according to claim 44 under conditions whereby said conjugatedisseminates through the circulatory system, and subjecting said patientto a planar or computer assisted tomographic nuclear medicine scan tovisualize a clot if present.